Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii.

Patchoulol, a plant-derived sesquiterpene compound, is widely used in perfumes, cosmetics, and pharmaceuticals. Microbial production provides a promising alternative approach for the efficient and sustainable production of patchoulol. However, there are no systematic engineering studies on Komagataella phaffii aimed at achieving high-yield patchoulol production. Herein, by fusion overexpression of FPP synthase and patchoulol synthase (ERG20LPTS), increasing the precursor supply, adjusting the copy number of ERG20LPTS and PTS, and combined with adding auxiliary carbon source and methanol concentration optimization, we constructed a high-yield patchoulol-producing strain P6H53, which produced 149.64 mg/L patchoulol in shake-flask fermentation with methanol as the substrate. In fed-batch fermentation, strain P6H53 achieved the highest production (2.47 g/L, 21.48 mg/g DCW, and 283.25 mg/L/d) to date in a 5 L fermenter. This study will lay a good foundation for the development of K. phaffii as a promising chassis microbial cell for the synthesis of patchoulol and other sesquiterpenes with methanol as the carbon source.

The in vitro fermentation of compound oral liquid by human colonic microbiota altered the abundance of probiotics and short-chain fatty acid production.

Compound oral liquid (COL), made from functional herbal foods, has gained immense popularity in China for healthcare. However, the interaction between the nutrients in COL and gut microbiota is still unclear. In our study, the content of total flavonoids, polyphenols, and proteins was increased and the total sugar reduced by crushing raw ingredients to 10 mesh (COL-C). After 24 h incubation with supplemented COL by human gut microbiota, the results of 16S rRNA high-throughput sequencing revealed that Faecalibacterium, Collinsella, Bifidobacterium, Megamonas, Lactobacillus, Phascolarctobacterium, and Dialister were enriched by COL. In particular, the latter three genera were observed to be significantly enriched after incubation with COL-C. Meanwhile, the abundance of Dorea, Clostridium XIVa, and Escherichia/Shigella was inhibited by COL. Moreover, the increased levels of acetate, propionate, and butyrate in COL were jointly contributed by supplementary carbohydrates and the enrichment of short-chain fatty acid (SCFA)-producing bacteria. In summary, our results indicated that the optimized extraction facilitated the nutrients to be dissolved out and enhanced the potential prebiotic effects for promoting the abundance of probiotics, suggesting that the nutrients in COL-C might improve the microbial structure by strengthening the metabolism of beneficial bacteria and restricting the conditioned pathogens more efficiently.

Association of human gut microbiota composition and metabolic functions with Ficus hirta Vahl dietary supplementation.

Ficus hirta Vahl (FHV), a traditional herbal ingredient of the tonic diet, receives increasing popularity in southern China. However, it is largely unknown that how a FHV diet (FHVD) affects the human gut microbiome. In this exploratory study, a total of 43 healthy individuals were randomized into the FHVD (n = 25) and Control (n = 18) groups to receive diet intervention for 8 weeks. 16S rRNA gene sequencing, metagenomic sequencing and metabolic profile of participants were measured to assess the association between FHV diet and gut microbiome. A preservation effect of Faecalibacterium and enrichment of Dialister, Veillonella, Clostridium, and Lachnospiraceae were found during the FHVD. Accordingly, the pathway of amino acid synthesis, citrate cycle, coenzyme synthesis, and partial B vitamin synthesis were found to be more abundant in the FHVD. In addition, serine, glutamine, gamma-aminobutyric acid, tryptamine, and short-chain fatty acids (SCFAs) were higher after the FHVD. The conjoint analysis of FHV components and in-vitro fermentation confirmed that the improved SCFAs concentration was collectively contributed by the increasing abundance of key enzyme genes and available substrates. In conclusion, the muti-omics analysis showed that the FHVD optimized the structure of the gut microbial community and its metabolic profile, leading to a healthy tendency, with a small cluster of bacteria driving the variation rather than a single taxon.

Modulation of Gut Microbiota Composition and Short-Chain Fatty Acid Synthesis by Mogroside V in an In Vitro Incubation System.

Mogroside V (MV), a sweetener, is one of the major components inSiraitia grosvenorii. In our research, after in vitro incubation with MV for 24 h, the human gut microbiota diversity changed, with an enrichment of the genera Bacteroides, Lactobacillus, Prevotella, Megasphaera, and Olsenella and the inhibition of Clostridium XlVa, Dorea, and Desulfovibrio. Moreover, the synthesis of short-chain fatty acids, such as acetate, propionate, and butyrate, was increased by gut microbiota. According to ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) analysis, MV was decomposed into secondary mogrosides, such as mogroside II/I and mogrol, by gut microbiota. Enhanced antioxidant abilities of the metabolites were found in the broth. The results suggested that MV, as a potential prebiotic, could benefit human health through its interaction with gut microbiota.

Application of RecET-Cre/loxP system in Corynebacterium glutamicum ATCC14067 for L-leucine production.

OBJECTIVE: To explore the RecET-Cre/loxP system for chromosomal replacement of promoter and its application on enhancement L-leucine production in Corynebacterium glutamicum (C. glutamicum) ATCC14067. RESULTS:  The RecET-Cre/loxP system was used to achieve the chromosomal replacement of promoter in C. glutamicum ATCC14067 to adjust the metabolic flux involving the L-leucine synthetic pathway. First, leuAr_13032 from C. glutamicum ATCC13032 which carried two mutations was overexpressed to release enzyme feedback inhibition. Then, comparing different mutations in ilvBNC gene clusters, the results indicated that ilvBNC_CP was most effective to enhance the metabolic flux of pyruvate towards L-leucine synthesis. The promoters of pck, odx and pyk2 were overexpressed under the strong promoter Peftu or Psod to improve the supply of pyruvate. Besides, the promoter PilvBNC was employed to dynamically control the transcription level of icd due to its attenuation mechanism by responding to the concentration of L-leucine. The final engineered strain produced 14.05 g L-leucine/L in flask cultivation. CONCLUSION:  The RecET-Cre/loxP system is effective for gene manipulation in C. glutamicum ATCC14067. Besides, the results demonstrate the potential of C. glutamicum ATCC14067 for L-leucine production and provide new targets and strategies for strain development.

Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

Synthetic biology approaches to access renewable carbon source utilization in Corynebacterium glutamicum.

Corynebacterium glutamicum (C. glutamicum), an important industrial workhorse, is capable of efficiently producing a variety of value-added chemicals and fuels beyond amino acids. C. glutamicum has a broad natural substrate spectrum and can simultaneously utilize various carbon sources in blends. The substrate spectrum of C. glutamicum has been further extended by detailed knowledge of carbon core metabolism and well-established genetic tools and engineering strategies. At present, many pathways have been successfully engineered in C. glutamicum for access to alternative renewable sources to produce natural or non-natural products, making C. glutamicum a promising and favorable microbial cell factory. In this review, we mainly focus on synthetic biology and metabolic engineering strategies for developing synthetic strains that grow on renewable sources to produce the target products. At the same time, we also explore the promotion and future challenges of existing synthetic biology platforms for industrial platform microorganism metabolic engineering efforts.